1. Field
The present disclosure relates to the covalent cross-linking of nucleic acid probes to target nucleic acids.
2. Background
Molecules that selectively bind to nucleic acids have a large variety of uses, including serving as indicators for the presence or absence of a specific nucleic acid in a sample, as well as being used to modify gene regulation and/or protein expression.
One example of an indicator role is in the technique of in situ hybridization, which allows for the detailed spatial and temporal mapping of nucleic acid sequences, such as mRNAs, in normal and pathological tissues. In situ hybridization can be used to study gene expression and regulation in a morphological context from the sub-cellular to the organismal levels.
Methods for regulating protein expression, including RNA interference (RNAi) and anti-sense, not only have great therapeutic potential, but also provide critical tools for biologists working to infer regulatory relationships from the phenotypes resulting from the knockdown of specific genes. RNA interference can be activated by exogenous small interfering RNAs (siRNAs) or short hairpin RNAs (shRNAs) that work in concert with endogenous protein enzymes to cleave and degrade the targeted mRNA transcript. By contrast, antisense methods employ base-pairing between an exogenous antisense RNA and the target mRNA transcript to downregulate translation via RNase H activation or steric interference. Both techniques are powerful and widely used but have significant limitations. Effectiveness and specificity vary significantly from one target sequence to another.
In some situations, one fundamental conceptual weakness shared by current methods utilizing nucleic acid binding molecules is the reliance on base pairing to provide both sequence specificity and binding affinity. In in situ hybridization methods, increasing the probe length to improve binding affinity for cognate targets (reducing false negatives during the wash step) simultaneously increases the opportunity for partial base-pairing to non-cognate targets (increasing false positives). In RNAi, the recognition site is fixed at approximately 21 base pairs, leading to variable effectiveness and specificity depending on the degree of competing native secondary structure in the mRNA, the degree of partial complementarity to non-cognate targets, and other unknown factors. In antisense, increasing the length of the recognition site improves affinity for the cognate target but also increases the likelihood of base-pairing to non-cognate targets.